Prestin Gene-Related Human Deafness Disease Animal Model Is Established

The hearing of mammal is very sensitivive, because there is a bio-amplifiers in the outer hair cells (OHC) of cochlea. It can amplify the movement to its 100 times in basement membrance via oscillations of outer hair cells body, so the sound can be heared. Oscillations of outer hair cells body is drived by a motor protein. Over the years, researchers have been looking for the motor protein that can cause oscillations of outer hair cells body. The prestin gene was cloned at the year 2000. Gene knock-out studies have shown that prestin is the motor protein in outer hair cells of inner ear. Prestin plays a role in hearing amplifier, in which it is closely related to human diseases of desfness. Two prestin point mutants have been found, which cause deafness. But we have not known the mechanism how prestin mutant causes deafness.It has been reported that prestin mutation in human can lead to deafness. In 2003, a point mutant, IVS2-2A/G, was found between intron 2 and exon 3. The point is located at the area of mRNA cut. Another mutant, R150Q, was found in exon 6 in 2007. Both the mutants cause hearing loss.Deafness is a disease with relatively high incidency, so its study is very important. We constructed a mouse model with knock-in technique, which analogs precisely human deafness caused by point mutation in exon 6 of prestin. We analyzed the phenotype of mutant mice, which is a convenient animal species to launch our studies. As a targeting point, we selected the exon 6 to find more detail.Methods:First, we designed and made a vector for gene tageting. Second, the vector which was linearized was electropotated into embryonic stem (ES) cells and the cells were cultured in G418 medium. We collected the cells when the numble of cells was coloned to a certain extent. Third, we extracted the genomic DNA, then the primer that designed could been used to select the colon by polymerase chain reaction (PCR). If homologous recombination succeeded, the specific band would appeared by PCR. Fourth, we injected the cells which were selected colon into blastula by microinjection, so we would obtain the chimeric mice. Fifth, we tried to identify germline transmission by hybrid. Last, we got homozygotes from the germline mice, then we analyzed in physiology, biochemical, protein and so on.The result showed that we made the vector of prestin knock-in, then transfected the ES cells. We gained the positive colon by G418. We injected the ES cells into blastocysts by micromanipulator and we obtained high-chimeric mice. Our work provided a clear direction for further elucidate the molecular mechanism of OHC.